Plasma Pyrophosphate Quantification

Blood was collected in 4.5 mL CTAD vacutainers (BD, 0.11 M buffered trisodium citrate solution, 15 M theophylline, 3.7 M adenosine and 0.198 M dipyridamole). 50 µL of a 15% K3-ethylenediaminetetraacetic acid (EDTA) solution was added and the tubes were centrifuged for 15 min (1000× g, 4 °C). Blood plasma was transferred to a Centrisart ultrafiltration tube (300 kDa, Sartorius, Göttingen, Germany), centrifuged for 30 min (2200× g, 4 °C) and the filtered plasma was stored at −80 °C until PP_i analysis. Plasma PP_i concentrations were determined as previously described [2 (link)]. In short, PP_i was converted into ATP using ATP sulfurylase in the presence of excess adenosine 5′-phosphosulfate (APS). For each 10 μL of plasma, 70 μL of a mixture containing 32 mU ATP sulfurylase (ENZ-353; ProSpec, East Brunswick, NJ, USA), 16 μmol/L APS (A5508; Sigma-Aldrich, St. Louis, MO, USA), 80 μmol/L MgCl₂, and 50 mmol/L HEPES (pH 7.4) was added. Following incubation at 37 °C for 30 min, ATP sulfurylase was inactivated at 90 °C for 10 min. Generated ATP was quantified using the ATP-monitoring reagent BacTiter-Glo (G8230; Promega, San Luis Obispo, CA, USA). An equal volume of BacTiter-Glo reagent was added to the samples. Bioluminescence was determined in a microplate reader (EnSpire Multimode Reader; PerkinElmer, Waltham, MA, USA). Each sample was measured twice in triplicates.